- Email: [email protected]
The nanomechanical role of melanin granules in the retinal pigment epithelium
NANO-01480; No of Page 1
Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx – xxx nanomedjournal.com
The nanomechanical role of melanin granules in the retinal pigment epithelium
1Q1 2
F
4
Michal Sarna, PhD a, b,⁎, Magdalena Olchawa, PhD a , Andrzej Zadlo, PhD a , Dawid Wnuk, MSc c , Tadeusz Sarna, PhD, DSc a
R O O
3Q2
a
5
b
6 7
8
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Poland Laboratory of Imaging and Atomic Force Spectroscopy, Malopolska Centre of Biotechnology, Jagiellonian University, Poland c Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Poland Received 29 June 2016; accepted 29 November 2016
Abstract
10
18
Nanomechanical properties of cells and tissues, in particular their elasticity, play an important role in different physiological and pathological processes. Recently, we have demonstrated that melanin granules dramatically modify nanomechanical properties of melanoma cells making them very stiff and, as a result, less aggressive. Although the mechanical effect of melanin granules was demonstrated in pathological cells, it was never studied in the case of normal cells. In this work, we analyzed the impact of melanin granules on nanomechanical properties of primary retinal pigment epithelium tissue fragments isolated from porcine eyes. The obtained results clearly show that melanin granules are responsible for the exceptional nanomechanical properties of the tissue. Our findings suggest that melanin granules in the retinal pigment epithelium may play an important role in sustaining the stiffness of this single cell layer, which functions as a natural mechanical barrier separating the retina from the choroid. © 2016 Published by Elsevier Inc.
19
Key words: Nanomechanical properties; Elasticity; Atomic force microscopy; Retinal pigment epithelium; Melanin granules; Blood-retinal barrier
15 16 17
D
E
14
T
13
C
12
E
11
P
9
26 27 28 29 30
R
25
O
24
C
23
Retinal pigment epithelium (RPE), a single layer of cells containing the pigment melanin, has several functions, such as light absorption, epithelial transport, spatial ion buffering, visual cycle, phagocytosis, secretion and immune modulation that are of key importance for the survival and proper function of the photoreceptor cells. 1 In pigmented tissues, including the RPE, melanin appears in the form of melanosomes – organelles displaying a range of different shapes and sizes. 2 Interestingly, melanin granules were found to have unusual nanomechanical properties being very stiff and hard to deform. 3 It should be
N
22
Funding organizations: This work was supported by a grant from the Polish National Science Centre (Maestro-2013/08/A/NZ1/00194). Confocal microscopy analysis was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract No. POIG.02.01.00-12-167/08, project Malopolska Centre of Biotechnology). The authors declare no conflict of interest. ⁎Corresponding author at: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Poland. E-mail address: [email protected] (M. Sarna).
U
21
R
20
emphasized that nanomechanical properties of cells and tissues play an important role in different physiological processes 4,5 and in certain pathologies. 6,7 Recently, we have shown that the presence of stiff melanin granules in melanoma cells dramatically modifies elastic properties of the cells, 8,9 making them less aggressive. 10 Although the nanomechanical role of melanin granules have been clearly shown in pathological cells, it has never been demonstrated in the case of normal cells. In normal tissues, melanin presence is most apparent in the skin, where it's content can vary significantly and strongly depends on racial and environmental factors. 11 Moreover, melanin pigmentation in the skin is subjected to continuous metabolisms. This makes it very difficult to examine the mechanical role of melanin granules in the epidermis. On the other hand, melanin content in the retinal pigment epithelium is independent of racial and environmental factors and shows little metabolic turnover after being synthesized during fetal development. 12 Although the main biological function of melanin in pigmented tissues is related to photoprotection, 13 the role of melanin in the RPE is still under extensive scrutiny. It is generally believed that melanin contributes to visual acuity by
http://dx.doi.org/10.1016/j.nano.2016.11.020 1549-9634/© 2016 Published by Elsevier Inc. Please cite this article as: Sarna M., et al., The nanomechanical role of melanin granules in the retinal pigment epithelium. Nanomedicine: NBM 2016;xx:06, http://dx.doi.org/10.1016/j.nano.2016.11.020
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
2
58 59 60 61 62 63 64 65 66 67 68 69 70 71
Methods
73
Primary Retinal Pigment Epithelium Tissue Fragments
74
84
RPE tissue fragments were isolated from porcine eyes based on protocols established for the isolation of human RPE as described elsewhere. 17 In brief, eyecups were prepared by dissecting the anterior segments and removing the vitreous and retina to expose the RPE monolayer. The RPE fragments were scraped from eyecups using a surgical scalpel and placed in multi-well plates containing glass coverslips. Samples were then incubated for 24 hours in MEM culture medium supplemented with 10% fetal bovine serum (FBS) and antibiotics to ensure that the fragments adhere tightly to glass coverslips for AFM analysis.
85
Melanin Isolation and Determination
86
For melanosome isolation, RPE cells were homogenized in phosphate buffered solution (PBS) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). Granules were then purified by ultracentrifugation in a discontinuous sucrose density gradient, according to the protocols described elsewhere. 18,19 Purified melanosome fraction, identified as a black pellet at the bottom of the centrifuge tube, was washed and resuspended in a small amount of PBS (pH 7.4). Determination of melanin in the cell samples was made using electron paramagnetic resonance (EPR) spectroscopy. 20 EPR was used because of high specificity of the technique in melanin detection and characterization. Other advantages of EPR, compared to alternative methods, such as photometric analysis, are nondestructive character of the technique and its ability to measure melanin in complex systems with high selectivity and sensitivity. This makes EPR a method of choice for melanin determination. For EPR analysis, 10 9 granules were suspended in PBS, frozen in liquid nitrogen and stored at 77 K. EPR measurements, were carried out in liquid nitrogen, using a standard fingertip quartz dewar and EMX-AA
81 82 83
87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
E
80
R
79
R
78
N C O
77
U
76
C
72
75
Atomic Force Microscopy
109
AFM analysis was conducted using a Bioscope Catalyst AFM (Bruker) coupled with an inverted optical microscope AxioObserver Z1 (Zeiss). RPE tissue fragments were analyzed in culture medium at 37 °C. AFM images of the tissue fragments were made using PeakForce Tapping mode. The use of this mode allowed better control of the force exerted on the cells, which is crucial when examining delicate biological samples. The employed mode also enabled high amplitude of cantilever oscillation, which is important when examining samples with high roughness such as tissue fragments. In addition, the PeakForce Capture was turned on, which resulted in acquiring a force-curve in each pixel of an image. For AFM imaging of RPE tissue fragments, a relatively soft cantilever was used with a nominal tip radius of 20 nm and with experimentally determined spring constant of 0.68 N/m (Bruker Probes). AFM analysis of melanosomes was performed on purified, unfixed melanin granules adsorbed onto freshly cleaved mica surface. It should be emphasized that melanin granules are too small for optical microscopy analysis due to limited resolution of the technique. On the other hand, electron microscopy, which is often used, requires the sample to be fixed, dried, covered with metal coating, and for transmission electron microscopy, cut into thin slices. Since AFM analysis can be performed in liquid environment and requires virtually no sample preparation, this method was found ideal for melanosome examination. Images of melanosomes were obtained in Tapping AC mode in PBS buffer at room temperature. Nanomechanical analysis of cells and melanosomes was made in force spectroscopy mode, which consisted of measuring force-displacement curves. In the case of cells, 20–30 force curves were taken from a single cell, which was selected using an optical microscope. 40 pigmented and 40 non-pigmented cells were analyzed. In the case of melanosomes, an AFM image was first acquired to precisely position the AFM tip on top of the granule. Then 5–10 force curves were collected from individual granules. A total number of 20 melanosomes were analyzed. For cells, a soft cantilever was used with a nominal tip radius of 20 nm and with experimentally determined spring constant of 0.01 N/m, whereas for melanosomes, a stiff cantilever with a nominal tip radius of 10 nm and with a spring constant determined to be 45 N/m was chosen (Bruker Probes). Spring constants of the used cantilevers were determined based on the thermal tune procedure as described elsewhere. 22 Data analysis of the obtained force-curves from both PeakForce Tapping and force spectroscopy was performed using AtomicJ software. In brief, force-displacement curves were converted into force-indentation curves and fitted with an appropriate model. In the case of cells, where indentation was large, the Sneddon model was used, whereas in the case of melanosomes in which low indentation was obtained the Hertz model was employed. Detailed information on the analysis of force curves can be found elsewhere. 23
110
F
57
R O O
56
105
P
55
spectrometer (Bruker BioSpin) operating at X-band with 100 kHz magnetic field modulation. Synthetic L-Dopa at a concentration of 0.57 mg/ml was used as standard. Detailed description of EPR analysis can be found elsewhere. 21
D
54
preventing light reflection from the fundus that may otherwise give rise to spurious signals, and protects the retina against light-induced oxidative stress. 14 However, other important functions such as mechanical stabilization of the RPE should also be considered. Importantly, RPE is part of the blood-retinal barrier (BRB) that separates the retina from the choroid. 15 The breakdown of the BRB has severe consequences for proper function of the posterior segments of the eye and occurs in several pathological conditions such as mechanical disruption, hydrostatic factors, metabolic diseases, inflammation and age-related macular degeneration. 16 This points to the unexplored nanomechanical properties of RPE, particularly the role of melanin granules in sustaining the stiffness of the BRB tissue. We address this issue in an ex vivo study of primary retinal pigment epithelium tissue fragments isolated from porcine eyes by analyzing the elastic properties of RPE cells employing atomic force microscopy and spectroscopy (AFM/S) technique. Our findings demonstrate that melanin granules have an important impact on the nanomechanical properties of the RPE tissue.
E
53
T
52
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
106 107 108
111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160
3
D
P
R O O
F
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
E
Figure 1. Melanin determination and nanomechanical characteristics of primary RPE cells. EPR spectra of isolated melanin granules (A) and synthetic L-dopa (B) used as standard. Histograms of the Young's modulus values for pigmented (C) and non-pigmented (D) RPE cells with log-normal fit to the data.
162
171
Analysis of the cells cytoskeleton was made on samples fixed with 3.7% formaldehyde, permeabilised with 0.1% Triton X-100 and blocked with 1% bovine serum albumin at room temperature. RPE tissue fragments were immunostained with mouse monoclonal anti-human α-tubulin IgG (Sigma-Aldrich) and Alexa Fluor 488-conjugated goat anti-mouse IgG (A110011, Life Technologies), and counterstained with Alexa Fluor 568-phalloidin (Life Technologies) and Hoechst 33,342 dye for DNA staining (Sigma-Aldrich). Images were obtained using a scanning laser confocal microscope (LSM 880, Zeiss).
172
Statistical Analysis
173
176
Statistical significance of differences in mean values was assessed using two-sample independent Student's t-test at the 95% confidence level. Statistical analysis was made using Mathematica 8.0 software.
177
Results
178
Figure 1 shows EPR spectra of melanosomes isolated from RPE cells (Figure 2, A) and of synthetic Dopa-melanin (Figure 2, B) used as a standard. The obtained EPR signal of the melanosomes is typical for the brown-black eumelanin. 24 This is consistent with EPR analysis of human RPE cells, which predominantly contains eumelanin. 25 Figure 1 also shows histograms of the Young's modulus (E) values, obtained by AFS, for pigmented (Figure 1, A) and non-pigmented (Figure 1,
167 168 169 170
174 175
179 180 181 182 183 184 185
E
R
166
R
165
N C O
164
U
163
B) RPE cells, respectively. Statistical analysis of the data revealed that the average E value (mean ± s.d.) for pigmented cells was 16.01 ± 0.45 kPa and 4.98 ± 0.17 kPa for non-pigmented cells. The difference in the mean values was statistically significant at a confidence level P b 0.0001. The distribution of the obtained E values for pigmented cells was much higher than that for non-pigmented cells and ranged from 1.55 to 97.91 kPa for pigmented cells and from 0.92 to 19.93 kPa for non-pigmented cells. The data clearly indicate that cells containing melanin granules were much stiffer then cells without the pigment. Figure 2 shows representative optical and AFM images of the morphology and nanomechanical properties of pigmented and non-pigmented RPE tissue fragments. Figure 2, A and B show bright field optical microscopy images of pigmented and non-pigmented RPE tissue fragments, respectively. The dark spots in the images correspond to melanin granules inside the cells. Figure 2, C-F show high resolution AFM images of the RPE cells. In the case of non-pigmented cells, actin filaments were easily seen all over the cell bodies, whereas in pigmented cells very few actin filaments were apparent only at the cell edges. Moreover, magnified images of the cells (Figure 2E and F) also revealed abundant microtubules in the case of non-pigmented cells. On the other hand, in pigmented cells, only microtubules above the nucleus were observed. In such cells, melanin granules were also clearly seen. These images indicate significant differences in the morphology between pigmented and non-pigmented cells with the most prominent being the absence of actin filaments in pigmented cells.
T
Immunofluorescence Analysis
C
161
186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
C
T
E
D
P
R O O
F
4
217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233
To determine whether the observed differences were caused by different arrangement of the cell cytoskeleton, scanning laser confocal microscopy (SLCM) was employed. Figure 3 shows representative confocal microscopy images taken from three different focusing levels of the cells. Hence, actin was taken at the bottom of the cells near the glass coverslip and in the middle of the cells, whereas microtubules were taken in the middle of the cells and at the top of the cells. Nuclei were taken correspondingly for both actin and microtubules positions. The images clearly show that actin was incorporated into thick filaments at the bottom of the cells near the glass coverslips, whereas in the middle of the cells actin was incorporated into thinner filaments, which were located in the cortex of the cells near the membrane. Microtubules, on the other hand, extended from the middle of the cells to the highest point of the cells. Based on confocal microscopy images we also determined that melanin granules were mostly localized in the middle of the cells above actin filaments and in between microtubules. SLCM data revealed no
U
216
N C O
R
R
E
Figure 2. Effect of melanin granules on the morphology and nanomechanical properties of RPE cells. Bright field optical images of pigmented (A) and non-pigmented (B) RPE tissue fragments. Red dotted squares indicate scanning areas covered with AFM. Scale bars in the images represent 40 μm. PeakForce error images of pigmented (C, D) and non-pigmented (E, F) tissue fragments. D and F show high magnification of the areas marked with white dotted squares in C and E. Scale bars in C and E represent 20 μm, whereas in D and F represent 5 μm. Corresponding force maps of the Young's modulus values for pigmented (G, H) and non-pigmented (I, J) RPE cells. H and J show high magnification of the areas marked with white dotted squares in G and I. Color bars in force maps indicate values of the Young's modulus ranging from 0 to 100 kPa (dark-to-bright) for G and H and from 0 to 20 kPa (dark-to-bright) for I and J. Arrows indicate actin filaments under the membrane in the cell cortex, arrow heads indicate microtubules whereas sharpened arrow heads indicate melanin granules.
significant differences in the cytoskeleton organization between pigmented and non-pigmented cells. It should be emphasized that AFM images of pigmented and non-pigmented cells, were obtained under identical experimental conditions with the same values of the set point, i.e. the force exerted on the cells by the AFM probe was the same for both pigmented and non-pigmented cells. The observation of distinct actin filaments in non-pigmented cells confirms that the cells were very soft and that the AFM probe before reaching the preset value of the set point deformed the cells to such an extent at which thick actin filaments in the bottom of the cells became visible. On the other hand, in pigmented cells, melanin granules located above actin filaments between microtubules, did not allow the AFM probe to deform the cells to the extent at which these filament could be seen. Only filaments at the cell edges could be seen in the case of pigmented cells. Exemplary force curves obtained during PeakForce Tapping (Fig. S1) clearly show that the deformation of pigmented cells during AFM
234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251
5
R
E
C
T
E
D
P
R O O
F
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
253 254 255 256 257 258 259 260 261 262 263 264 265 266 267
imaging was much smaller than that of non-pigmented cells. That is why in pigmented cells actin filaments at the bottom of the cells were not observed. In addition, pigmented cells appeared to be much higher and more rounded than non-pigmented cells, which looked flat and rough. This was confirmed by comparing the topographical cross sections of the cells (Fig. S2). Indeed pigmented cells were significantly higher and smoother than non-pigmented cells. Corresponding force maps of the cells, shown in Figure 2, G– J, clearly demonstrate that melanin granules were responsible for higher values of the Young's modules of pigmented RPE cells. It should be emphasized that the color scales in the force maps were extended to maximum in both pigmented and non-pigmented images to better highlight the image features. Notably, in pigmented cells only melanin granules are visible, whereas in non-pigmented cells both actin filaments and
U
252
N C O
R
Figure 3. Cytoskeleton organization in pigmented and non-pigmented RPE cells. Transmitted laser light images of pigmented (A) and non-pigmented (B) RPE tissue fragments. The dark color in the images indicate melanin granules inside the cells. Combined fluorescence images of actin (red) and nuclei (blue) at two different focusing levels for pigmented (C, D) and non-pigmented (E, F) cells followed by combined fluorescence images of microtubules (green) and nuclei (blue) at two different focusing levels for pigmented (G, H) and non-pigmented (I, J) cells. Arrows indicate actin filaments under the membrane in the cell cortex, whereas arrow heads indicate microtubules above the nucleus. Scale bars for all images represent 20 μm.
microtubules are seen. This indicates that the Young's modulus values of the pigment granules are much greater than those of individual actin filaments and microtubules. Elasticity analysis of isolated melanosomes confirmed that the pigment granules were very stiff and hard to deform. Representative AFM image of isolated melanin granules is shown in Figure 4, A, while Figure 4, B shows a histogram of the Young's modulus values obtained for the analyzed melanosomes. Statistical analysis revealed that the average value of the Young's modulus for the pigment granules (mean ± s.d.) was 4.49 ± 0.11 MPa. These results are consistent with elastic properties of isolated human ocular melanosomes, which were found to have an average value of the Young's modulus of the order of MPa. 4 It should be emphasized that the Young's modulus values for the pigment granules are much greater than those of any other organelles found in cells. The results shed new
268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
R O O
F
6
Figure 4. Atomic force microscopy analysis of isolated RPE melanosomes. Amplitude image of melanin granules (A). Inset shows a magnified view of an individual granule marked with white dotted square. Scale bar represents 500 nm. Histogram of the Young's modulus values for the pigment granules with Gaussian fit to the data (B).
288 289 290 291
Discussion
293
In this work we have demonstrated that melanin granules are responsible for the exceptional nanomechanical properties of the RPE tissue. The inherent heterogeneity of melanin content observed in RPE cells, 29 allowed us to compare the elasticity between pigmented and non-pigmented cells from the same tissue fragments. These results provide new inside look at the nanomechanical role of melanin granules in the RPE tissue. According to the current paradigm, it is the Bruch's membrane (BM), a specialized basement membrane beneath the RPE layer that plays a major role as a mechanical barrier. 30 Results obtained in this work clearly show that the values of the Young's modulus for RPE are much greater than those reported for BM. 31 Based on results of our present study, we can conclude that RPE, rather than BM, is a major contributor to the mechanical barrier of the RPE-choroid complex. In a concurrent study we demonstrated that prolonged irradiation of isolated porcine and bovine melanin granules with intense visible light – an in vitro model for photo-aging of RPE melanosomes, 32 brought about a substantial softening of the pigment granules. Moreover, RPE cells with phagocytized photo-bleached melanosomes, appeared to be much softer than cells with untreated melanin granules (data not published). These results suggest that age-related changes in the nanomechanical properties of RPE melanosomes may correlate with the development of certain degenerative diseases such as age-related macular degeneration (AMD). Notably, one of the clinical symptoms of the so called ‘wet type’ AMD is mechanical breach of the RPE and abnormal growth of new blood vessels
299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320
E
R
298
R
297
N C O
296
U
295
C
292
294
P
287
D
286
E
285
into the retina, which leads to gradual loss of vision. 33 A very interesting study was recently published, in which multispectral imaging was employed to view RPE melanosomes in patients diagnosed with AMD. 34 The data suggested a clear relationship between severity of disease and the degree of melanin disruption. Therefore the reported effect of mechanical inhibition on angiogenesis during the surgical treatment of varicose veins, 35 is of considerable interest for it supports the postulated by us role of RPE as a mechanical barrier.
light on cellular mechanics of pigmented cells. Until now it was believed that all major factors responsible for the mechanical properties of cells were well understood. Thus, the main contributor to the cell stiffness supposed to be the cytoskeleton, 26 in particular actin and intermittent filaments. 27,28 Our findings show that in pigmented cells, melanin granules have a much higher impact on nanomechanical properties of the cells than the cytoskeleton.
T
284
321 322 323 324 325 326 327 328 329
Appendix A. Supplementary data
330
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2016.11.020.
331
References
333
1. Strauss O. The retinal pigment epithelium in visual function. Physiol Rev 2005;85:845-81. 2. Liu Y, Hong L, Wakamatsu K, et al. Comparison of structural and chemical properties of black and red human hair melanosomes. Photochem Photobiol 2005;81(1):135-44. 3. Guo S, Hong L, Akhremitchev BB, Simon JD. Surface elastic properties of human retinal pigment epithelium melanosomes. Photochem Photobiol 2008;84(3):671-8. 4. Fels J, Jeggle P, Liashkovich I, Peters W, Oberleithner H. Nanomechanics of vascular endothelium. Cell Tissue Res 2014;355(3):727-37. 5. Schimpel C, Werzer O, Fröhlich E, et al. Atomic force microscopy as analytical tool to study physico-mechanical properties of intestinal cells. Beilstein J Nanotechnol 2015;6:1457-66. 6. Cross SE, Jin YS, Rao J, Gimzewski JK. Nanomechanical analysis of cells from cancer patients. Nat Nanotechnol 2007;2(12):780-3. 7. Sarna M, Wojcik KA, Hermanowicz P, et al. Undifferentiated bronchial fibroblasts derived from asthmatic patients display higher elastic modulus than their non-asthmatic counterparts. PLoS One 2015;10(2):e0116840. 8. Sarna M, Zadlo A, Koczurkiewicz P, Burda K, Sarna T. Malanin Modifies Nanomechanical Properties of Melanoma Cells. The melanocyte and its environment. Bologna, Italy: Medimond; 2012. p. 23-8. 9. Sarna M, Zadlo A, Pilat A, et al. Nanomechanical analysis of pigmented human melanoma cells. Pigment Cell Melanoma Res 2013;26(5):727-30.
332
334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
E
D
P
R O O
F
23. Hermanowicz P, Sarna M, Burda K, Gabryś H. AtomicJ: an open source software for analysis of force curves. Rev Sci Instrum 2014;85(6):063703. 24. Sealy RC, Hyde JS, Felix CC, Menon IA, Prota G. Eumelanins and pheomelanins: characterization by electron spin resonance spectroscopy. Science 1982;217(4559):545-7. 25. Sarna T, Burke JM, Korytowski W, Rozanowska M, Skumatz CM, Zareba A, et al. Loss of melanin from human RPE with aging: possible role of melanin photooxidation. Exp Eye Res 2001;76:89-98. 26. Gefen A, Weihs D. Mechanical cytoprotection: a review of cytoskeletonprotection approaches for cells. J Biomech 2016;49(8):1321-9. 27. Vargas-Pinto R, Gong H, Vahabikashi A, Johnson M. The effect of the endothelial cell cortex on atomic force microscopy measurements. Biophys J 2013;105(2):300-9. 28. Wagner OI, Rammensee S, Korde N, Wen Q, Leterrier JF, Janmey PA. Softness, strength and self-repair in intermediate filament networks. Exp Cell Res 2007;313(10):2228-35. 29. Burke JM, Skumatz CM, Irving PE, McKay BS. Phenotypic heterogeneity of retinal pigment epithelial cells in vitro and in situ. Exp Eye Res 1996;62(1):63-73. 30. Booij JC, Baas DC, Beisekeeva J, Gorgels TG, Bergen AA. The dynamic nature of Bruch's membrane. Prog Retin Eye Res 2010;29(1):1-18. 31. Last JA, Liliensiek SJ, Nealey PF, Murphya CJ. Determining the mechanical properties of human corneal basement membranes with atomic force microscopy. Struct Biol 2009;167(1):19-24. 32. Zadlo A, Rozanowska MB, Burke JB, Sarna T. Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit ironinduced peroxidation of lipids. Pigment Cell Res 2007;20:52-60. 33. Luthert PJ. Pathogenesis of age-related macular degeneration. Diagn Histopathol 2011;17(1):10-6. 34. Dugel PU, Zimmer CN. Imaging of melanin disruption in age-related macular degeneration using multispectral imaging. Ophthalmic Surg Lasers Imaging Retina 2016;47(2):134-41. 35. van Rij AM, Jones GT, Hill BG, et al. Mechanical inhibition of angiogenesis at the saphenofemoral junction in the surgical treatment of varicose veins: early results of a blinded randomized controlled trial. Circulation 2008;118(1):66-74.
C
T
10. Sarna M, Zadlo A, Hermanowicz P, Madeja Z, Burda K, Sarna T. Cell elasticity is an important indicator of the metastatic phenotype of melanoma cells. Exp Dermatol 2014;23(11):813-8. 11. Lin JY, Fisher DE. Melanocyte biology and skin pigmentation. Nature 2007;445:843-50. 12. Boulton ME. Studying melanin and lipofuscin in RPE cell culture models. Exp Eye Res 2014;126:61-7. 13. Seagle BL, Rezai KA, Kobori Y, Gasyna EM, Rezaei KA, Norris JR. Melanin photoprotection in the human retinal pigment epithelium and its correlation with light-induced cell apoptosis. Proc Natl Acad Sci U S A 2005;102(25):8978-83. 14. Sarna T. Properties and function of the ocular melanin – a photobiophysical view. J Photochem Photobiol B 1992;12(3):215-58. 15. Campbell M, Humphries P. The blood-retina barrier: tight junctions and barrier modulation. Adv Exp Med Biol 2012;763:70-84. 16. Klaassen I, Van Noorden CJ, Schlingemann RO. Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions. Prog Retin Eye Res 2013;34:19-48. 17. Burke JM, Jaffe GJ, Brzeski CM. The effect of culture density and proliferation rate on the expression of ouabain-sensitive Na/K ATPase pumps in cultured human retinal pigment epithelium. Exp Cell Res 1991;194(2):190-4. 18. Burke JM, Henry MM, Zareba M, Sarna T. Photobleaching of melanosomes from retinal pigment epithelium: I. Effects on protein oxidation. Photochem Photobiol 2007;83(4):920-4. 19. Zareba M, Sarna T, Szewczyk G, Burke JM. Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress. Photochem Photobiol 2007;83(4):925-30. 20. d'Ischia M, Wakamatsu K, Napolitano A, et al. Melanins and melanogenesis: methods, standards, protocols. Pigment Cell Melanoma Res 2013;26(5):616-33. 21. Zadlo A, Burke JM, Sarna T. Effect of untreated and photobleached bovine RPE melanosomes on the photoinduced peroxidation of lipids. Photochem Photobiol Sci 2009;8(6):830-7. 22. Hutter JL, Bechhoefer J. Calibration of atomic-force microscope tips. Rev Sci Instrum 1993;64:1868.
E
358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394
U
N C O
R
R
433
7 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432
Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx – xxx nanomedjournal.com
The nanomechanical role of melanin granules in the retinal pigment epithelium
1Q1 2
F
4
Michal Sarna, PhD a, b,⁎, Magdalena Olchawa, PhD a , Andrzej Zadlo, PhD a , Dawid Wnuk, MSc c , Tadeusz Sarna, PhD, DSc a
R O O
3Q2
a
5
b
6 7
8
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Poland Laboratory of Imaging and Atomic Force Spectroscopy, Malopolska Centre of Biotechnology, Jagiellonian University, Poland c Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Poland Received 29 June 2016; accepted 29 November 2016
Abstract
10
18
Nanomechanical properties of cells and tissues, in particular their elasticity, play an important role in different physiological and pathological processes. Recently, we have demonstrated that melanin granules dramatically modify nanomechanical properties of melanoma cells making them very stiff and, as a result, less aggressive. Although the mechanical effect of melanin granules was demonstrated in pathological cells, it was never studied in the case of normal cells. In this work, we analyzed the impact of melanin granules on nanomechanical properties of primary retinal pigment epithelium tissue fragments isolated from porcine eyes. The obtained results clearly show that melanin granules are responsible for the exceptional nanomechanical properties of the tissue. Our findings suggest that melanin granules in the retinal pigment epithelium may play an important role in sustaining the stiffness of this single cell layer, which functions as a natural mechanical barrier separating the retina from the choroid. © 2016 Published by Elsevier Inc.
19
Key words: Nanomechanical properties; Elasticity; Atomic force microscopy; Retinal pigment epithelium; Melanin granules; Blood-retinal barrier
15 16 17
D
E
14
T
13
C
12
E
11
P
9
26 27 28 29 30
R
25
O
24
C
23
Retinal pigment epithelium (RPE), a single layer of cells containing the pigment melanin, has several functions, such as light absorption, epithelial transport, spatial ion buffering, visual cycle, phagocytosis, secretion and immune modulation that are of key importance for the survival and proper function of the photoreceptor cells. 1 In pigmented tissues, including the RPE, melanin appears in the form of melanosomes – organelles displaying a range of different shapes and sizes. 2 Interestingly, melanin granules were found to have unusual nanomechanical properties being very stiff and hard to deform. 3 It should be
N
22
Funding organizations: This work was supported by a grant from the Polish National Science Centre (Maestro-2013/08/A/NZ1/00194). Confocal microscopy analysis was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract No. POIG.02.01.00-12-167/08, project Malopolska Centre of Biotechnology). The authors declare no conflict of interest. ⁎Corresponding author at: Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Poland. E-mail address: [email protected] (M. Sarna).
U
21
R
20
emphasized that nanomechanical properties of cells and tissues play an important role in different physiological processes 4,5 and in certain pathologies. 6,7 Recently, we have shown that the presence of stiff melanin granules in melanoma cells dramatically modifies elastic properties of the cells, 8,9 making them less aggressive. 10 Although the nanomechanical role of melanin granules have been clearly shown in pathological cells, it has never been demonstrated in the case of normal cells. In normal tissues, melanin presence is most apparent in the skin, where it's content can vary significantly and strongly depends on racial and environmental factors. 11 Moreover, melanin pigmentation in the skin is subjected to continuous metabolisms. This makes it very difficult to examine the mechanical role of melanin granules in the epidermis. On the other hand, melanin content in the retinal pigment epithelium is independent of racial and environmental factors and shows little metabolic turnover after being synthesized during fetal development. 12 Although the main biological function of melanin in pigmented tissues is related to photoprotection, 13 the role of melanin in the RPE is still under extensive scrutiny. It is generally believed that melanin contributes to visual acuity by
http://dx.doi.org/10.1016/j.nano.2016.11.020 1549-9634/© 2016 Published by Elsevier Inc. Please cite this article as: Sarna M., et al., The nanomechanical role of melanin granules in the retinal pigment epithelium. Nanomedicine: NBM 2016;xx:06, http://dx.doi.org/10.1016/j.nano.2016.11.020
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
2
58 59 60 61 62 63 64 65 66 67 68 69 70 71
Methods
73
Primary Retinal Pigment Epithelium Tissue Fragments
74
84
RPE tissue fragments were isolated from porcine eyes based on protocols established for the isolation of human RPE as described elsewhere. 17 In brief, eyecups were prepared by dissecting the anterior segments and removing the vitreous and retina to expose the RPE monolayer. The RPE fragments were scraped from eyecups using a surgical scalpel and placed in multi-well plates containing glass coverslips. Samples were then incubated for 24 hours in MEM culture medium supplemented with 10% fetal bovine serum (FBS) and antibiotics to ensure that the fragments adhere tightly to glass coverslips for AFM analysis.
85
Melanin Isolation and Determination
86
For melanosome isolation, RPE cells were homogenized in phosphate buffered solution (PBS) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). Granules were then purified by ultracentrifugation in a discontinuous sucrose density gradient, according to the protocols described elsewhere. 18,19 Purified melanosome fraction, identified as a black pellet at the bottom of the centrifuge tube, was washed and resuspended in a small amount of PBS (pH 7.4). Determination of melanin in the cell samples was made using electron paramagnetic resonance (EPR) spectroscopy. 20 EPR was used because of high specificity of the technique in melanin detection and characterization. Other advantages of EPR, compared to alternative methods, such as photometric analysis, are nondestructive character of the technique and its ability to measure melanin in complex systems with high selectivity and sensitivity. This makes EPR a method of choice for melanin determination. For EPR analysis, 10 9 granules were suspended in PBS, frozen in liquid nitrogen and stored at 77 K. EPR measurements, were carried out in liquid nitrogen, using a standard fingertip quartz dewar and EMX-AA
81 82 83
87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
E
80
R
79
R
78
N C O
77
U
76
C
72
75
Atomic Force Microscopy
109
AFM analysis was conducted using a Bioscope Catalyst AFM (Bruker) coupled with an inverted optical microscope AxioObserver Z1 (Zeiss). RPE tissue fragments were analyzed in culture medium at 37 °C. AFM images of the tissue fragments were made using PeakForce Tapping mode. The use of this mode allowed better control of the force exerted on the cells, which is crucial when examining delicate biological samples. The employed mode also enabled high amplitude of cantilever oscillation, which is important when examining samples with high roughness such as tissue fragments. In addition, the PeakForce Capture was turned on, which resulted in acquiring a force-curve in each pixel of an image. For AFM imaging of RPE tissue fragments, a relatively soft cantilever was used with a nominal tip radius of 20 nm and with experimentally determined spring constant of 0.68 N/m (Bruker Probes). AFM analysis of melanosomes was performed on purified, unfixed melanin granules adsorbed onto freshly cleaved mica surface. It should be emphasized that melanin granules are too small for optical microscopy analysis due to limited resolution of the technique. On the other hand, electron microscopy, which is often used, requires the sample to be fixed, dried, covered with metal coating, and for transmission electron microscopy, cut into thin slices. Since AFM analysis can be performed in liquid environment and requires virtually no sample preparation, this method was found ideal for melanosome examination. Images of melanosomes were obtained in Tapping AC mode in PBS buffer at room temperature. Nanomechanical analysis of cells and melanosomes was made in force spectroscopy mode, which consisted of measuring force-displacement curves. In the case of cells, 20–30 force curves were taken from a single cell, which was selected using an optical microscope. 40 pigmented and 40 non-pigmented cells were analyzed. In the case of melanosomes, an AFM image was first acquired to precisely position the AFM tip on top of the granule. Then 5–10 force curves were collected from individual granules. A total number of 20 melanosomes were analyzed. For cells, a soft cantilever was used with a nominal tip radius of 20 nm and with experimentally determined spring constant of 0.01 N/m, whereas for melanosomes, a stiff cantilever with a nominal tip radius of 10 nm and with a spring constant determined to be 45 N/m was chosen (Bruker Probes). Spring constants of the used cantilevers were determined based on the thermal tune procedure as described elsewhere. 22 Data analysis of the obtained force-curves from both PeakForce Tapping and force spectroscopy was performed using AtomicJ software. In brief, force-displacement curves were converted into force-indentation curves and fitted with an appropriate model. In the case of cells, where indentation was large, the Sneddon model was used, whereas in the case of melanosomes in which low indentation was obtained the Hertz model was employed. Detailed information on the analysis of force curves can be found elsewhere. 23
110
F
57
R O O
56
105
P
55
spectrometer (Bruker BioSpin) operating at X-band with 100 kHz magnetic field modulation. Synthetic L-Dopa at a concentration of 0.57 mg/ml was used as standard. Detailed description of EPR analysis can be found elsewhere. 21
D
54
preventing light reflection from the fundus that may otherwise give rise to spurious signals, and protects the retina against light-induced oxidative stress. 14 However, other important functions such as mechanical stabilization of the RPE should also be considered. Importantly, RPE is part of the blood-retinal barrier (BRB) that separates the retina from the choroid. 15 The breakdown of the BRB has severe consequences for proper function of the posterior segments of the eye and occurs in several pathological conditions such as mechanical disruption, hydrostatic factors, metabolic diseases, inflammation and age-related macular degeneration. 16 This points to the unexplored nanomechanical properties of RPE, particularly the role of melanin granules in sustaining the stiffness of the BRB tissue. We address this issue in an ex vivo study of primary retinal pigment epithelium tissue fragments isolated from porcine eyes by analyzing the elastic properties of RPE cells employing atomic force microscopy and spectroscopy (AFM/S) technique. Our findings demonstrate that melanin granules have an important impact on the nanomechanical properties of the RPE tissue.
E
53
T
52
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
106 107 108
111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160
3
D
P
R O O
F
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
E
Figure 1. Melanin determination and nanomechanical characteristics of primary RPE cells. EPR spectra of isolated melanin granules (A) and synthetic L-dopa (B) used as standard. Histograms of the Young's modulus values for pigmented (C) and non-pigmented (D) RPE cells with log-normal fit to the data.
162
171
Analysis of the cells cytoskeleton was made on samples fixed with 3.7% formaldehyde, permeabilised with 0.1% Triton X-100 and blocked with 1% bovine serum albumin at room temperature. RPE tissue fragments were immunostained with mouse monoclonal anti-human α-tubulin IgG (Sigma-Aldrich) and Alexa Fluor 488-conjugated goat anti-mouse IgG (A110011, Life Technologies), and counterstained with Alexa Fluor 568-phalloidin (Life Technologies) and Hoechst 33,342 dye for DNA staining (Sigma-Aldrich). Images were obtained using a scanning laser confocal microscope (LSM 880, Zeiss).
172
Statistical Analysis
173
176
Statistical significance of differences in mean values was assessed using two-sample independent Student's t-test at the 95% confidence level. Statistical analysis was made using Mathematica 8.0 software.
177
Results
178
Figure 1 shows EPR spectra of melanosomes isolated from RPE cells (Figure 2, A) and of synthetic Dopa-melanin (Figure 2, B) used as a standard. The obtained EPR signal of the melanosomes is typical for the brown-black eumelanin. 24 This is consistent with EPR analysis of human RPE cells, which predominantly contains eumelanin. 25 Figure 1 also shows histograms of the Young's modulus (E) values, obtained by AFS, for pigmented (Figure 1, A) and non-pigmented (Figure 1,
167 168 169 170
174 175
179 180 181 182 183 184 185
E
R
166
R
165
N C O
164
U
163
B) RPE cells, respectively. Statistical analysis of the data revealed that the average E value (mean ± s.d.) for pigmented cells was 16.01 ± 0.45 kPa and 4.98 ± 0.17 kPa for non-pigmented cells. The difference in the mean values was statistically significant at a confidence level P b 0.0001. The distribution of the obtained E values for pigmented cells was much higher than that for non-pigmented cells and ranged from 1.55 to 97.91 kPa for pigmented cells and from 0.92 to 19.93 kPa for non-pigmented cells. The data clearly indicate that cells containing melanin granules were much stiffer then cells without the pigment. Figure 2 shows representative optical and AFM images of the morphology and nanomechanical properties of pigmented and non-pigmented RPE tissue fragments. Figure 2, A and B show bright field optical microscopy images of pigmented and non-pigmented RPE tissue fragments, respectively. The dark spots in the images correspond to melanin granules inside the cells. Figure 2, C-F show high resolution AFM images of the RPE cells. In the case of non-pigmented cells, actin filaments were easily seen all over the cell bodies, whereas in pigmented cells very few actin filaments were apparent only at the cell edges. Moreover, magnified images of the cells (Figure 2E and F) also revealed abundant microtubules in the case of non-pigmented cells. On the other hand, in pigmented cells, only microtubules above the nucleus were observed. In such cells, melanin granules were also clearly seen. These images indicate significant differences in the morphology between pigmented and non-pigmented cells with the most prominent being the absence of actin filaments in pigmented cells.
T
Immunofluorescence Analysis
C
161
186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
C
T
E
D
P
R O O
F
4
217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233
To determine whether the observed differences were caused by different arrangement of the cell cytoskeleton, scanning laser confocal microscopy (SLCM) was employed. Figure 3 shows representative confocal microscopy images taken from three different focusing levels of the cells. Hence, actin was taken at the bottom of the cells near the glass coverslip and in the middle of the cells, whereas microtubules were taken in the middle of the cells and at the top of the cells. Nuclei were taken correspondingly for both actin and microtubules positions. The images clearly show that actin was incorporated into thick filaments at the bottom of the cells near the glass coverslips, whereas in the middle of the cells actin was incorporated into thinner filaments, which were located in the cortex of the cells near the membrane. Microtubules, on the other hand, extended from the middle of the cells to the highest point of the cells. Based on confocal microscopy images we also determined that melanin granules were mostly localized in the middle of the cells above actin filaments and in between microtubules. SLCM data revealed no
U
216
N C O
R
R
E
Figure 2. Effect of melanin granules on the morphology and nanomechanical properties of RPE cells. Bright field optical images of pigmented (A) and non-pigmented (B) RPE tissue fragments. Red dotted squares indicate scanning areas covered with AFM. Scale bars in the images represent 40 μm. PeakForce error images of pigmented (C, D) and non-pigmented (E, F) tissue fragments. D and F show high magnification of the areas marked with white dotted squares in C and E. Scale bars in C and E represent 20 μm, whereas in D and F represent 5 μm. Corresponding force maps of the Young's modulus values for pigmented (G, H) and non-pigmented (I, J) RPE cells. H and J show high magnification of the areas marked with white dotted squares in G and I. Color bars in force maps indicate values of the Young's modulus ranging from 0 to 100 kPa (dark-to-bright) for G and H and from 0 to 20 kPa (dark-to-bright) for I and J. Arrows indicate actin filaments under the membrane in the cell cortex, arrow heads indicate microtubules whereas sharpened arrow heads indicate melanin granules.
significant differences in the cytoskeleton organization between pigmented and non-pigmented cells. It should be emphasized that AFM images of pigmented and non-pigmented cells, were obtained under identical experimental conditions with the same values of the set point, i.e. the force exerted on the cells by the AFM probe was the same for both pigmented and non-pigmented cells. The observation of distinct actin filaments in non-pigmented cells confirms that the cells were very soft and that the AFM probe before reaching the preset value of the set point deformed the cells to such an extent at which thick actin filaments in the bottom of the cells became visible. On the other hand, in pigmented cells, melanin granules located above actin filaments between microtubules, did not allow the AFM probe to deform the cells to the extent at which these filament could be seen. Only filaments at the cell edges could be seen in the case of pigmented cells. Exemplary force curves obtained during PeakForce Tapping (Fig. S1) clearly show that the deformation of pigmented cells during AFM
234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251
5
R
E
C
T
E
D
P
R O O
F
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
253 254 255 256 257 258 259 260 261 262 263 264 265 266 267
imaging was much smaller than that of non-pigmented cells. That is why in pigmented cells actin filaments at the bottom of the cells were not observed. In addition, pigmented cells appeared to be much higher and more rounded than non-pigmented cells, which looked flat and rough. This was confirmed by comparing the topographical cross sections of the cells (Fig. S2). Indeed pigmented cells were significantly higher and smoother than non-pigmented cells. Corresponding force maps of the cells, shown in Figure 2, G– J, clearly demonstrate that melanin granules were responsible for higher values of the Young's modules of pigmented RPE cells. It should be emphasized that the color scales in the force maps were extended to maximum in both pigmented and non-pigmented images to better highlight the image features. Notably, in pigmented cells only melanin granules are visible, whereas in non-pigmented cells both actin filaments and
U
252
N C O
R
Figure 3. Cytoskeleton organization in pigmented and non-pigmented RPE cells. Transmitted laser light images of pigmented (A) and non-pigmented (B) RPE tissue fragments. The dark color in the images indicate melanin granules inside the cells. Combined fluorescence images of actin (red) and nuclei (blue) at two different focusing levels for pigmented (C, D) and non-pigmented (E, F) cells followed by combined fluorescence images of microtubules (green) and nuclei (blue) at two different focusing levels for pigmented (G, H) and non-pigmented (I, J) cells. Arrows indicate actin filaments under the membrane in the cell cortex, whereas arrow heads indicate microtubules above the nucleus. Scale bars for all images represent 20 μm.
microtubules are seen. This indicates that the Young's modulus values of the pigment granules are much greater than those of individual actin filaments and microtubules. Elasticity analysis of isolated melanosomes confirmed that the pigment granules were very stiff and hard to deform. Representative AFM image of isolated melanin granules is shown in Figure 4, A, while Figure 4, B shows a histogram of the Young's modulus values obtained for the analyzed melanosomes. Statistical analysis revealed that the average value of the Young's modulus for the pigment granules (mean ± s.d.) was 4.49 ± 0.11 MPa. These results are consistent with elastic properties of isolated human ocular melanosomes, which were found to have an average value of the Young's modulus of the order of MPa. 4 It should be emphasized that the Young's modulus values for the pigment granules are much greater than those of any other organelles found in cells. The results shed new
268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
R O O
F
6
Figure 4. Atomic force microscopy analysis of isolated RPE melanosomes. Amplitude image of melanin granules (A). Inset shows a magnified view of an individual granule marked with white dotted square. Scale bar represents 500 nm. Histogram of the Young's modulus values for the pigment granules with Gaussian fit to the data (B).
288 289 290 291
Discussion
293
In this work we have demonstrated that melanin granules are responsible for the exceptional nanomechanical properties of the RPE tissue. The inherent heterogeneity of melanin content observed in RPE cells, 29 allowed us to compare the elasticity between pigmented and non-pigmented cells from the same tissue fragments. These results provide new inside look at the nanomechanical role of melanin granules in the RPE tissue. According to the current paradigm, it is the Bruch's membrane (BM), a specialized basement membrane beneath the RPE layer that plays a major role as a mechanical barrier. 30 Results obtained in this work clearly show that the values of the Young's modulus for RPE are much greater than those reported for BM. 31 Based on results of our present study, we can conclude that RPE, rather than BM, is a major contributor to the mechanical barrier of the RPE-choroid complex. In a concurrent study we demonstrated that prolonged irradiation of isolated porcine and bovine melanin granules with intense visible light – an in vitro model for photo-aging of RPE melanosomes, 32 brought about a substantial softening of the pigment granules. Moreover, RPE cells with phagocytized photo-bleached melanosomes, appeared to be much softer than cells with untreated melanin granules (data not published). These results suggest that age-related changes in the nanomechanical properties of RPE melanosomes may correlate with the development of certain degenerative diseases such as age-related macular degeneration (AMD). Notably, one of the clinical symptoms of the so called ‘wet type’ AMD is mechanical breach of the RPE and abnormal growth of new blood vessels
299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320
E
R
298
R
297
N C O
296
U
295
C
292
294
P
287
D
286
E
285
into the retina, which leads to gradual loss of vision. 33 A very interesting study was recently published, in which multispectral imaging was employed to view RPE melanosomes in patients diagnosed with AMD. 34 The data suggested a clear relationship between severity of disease and the degree of melanin disruption. Therefore the reported effect of mechanical inhibition on angiogenesis during the surgical treatment of varicose veins, 35 is of considerable interest for it supports the postulated by us role of RPE as a mechanical barrier.
light on cellular mechanics of pigmented cells. Until now it was believed that all major factors responsible for the mechanical properties of cells were well understood. Thus, the main contributor to the cell stiffness supposed to be the cytoskeleton, 26 in particular actin and intermittent filaments. 27,28 Our findings show that in pigmented cells, melanin granules have a much higher impact on nanomechanical properties of the cells than the cytoskeleton.
T
284
321 322 323 324 325 326 327 328 329
Appendix A. Supplementary data
330
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2016.11.020.
331
References
333
1. Strauss O. The retinal pigment epithelium in visual function. Physiol Rev 2005;85:845-81. 2. Liu Y, Hong L, Wakamatsu K, et al. Comparison of structural and chemical properties of black and red human hair melanosomes. Photochem Photobiol 2005;81(1):135-44. 3. Guo S, Hong L, Akhremitchev BB, Simon JD. Surface elastic properties of human retinal pigment epithelium melanosomes. Photochem Photobiol 2008;84(3):671-8. 4. Fels J, Jeggle P, Liashkovich I, Peters W, Oberleithner H. Nanomechanics of vascular endothelium. Cell Tissue Res 2014;355(3):727-37. 5. Schimpel C, Werzer O, Fröhlich E, et al. Atomic force microscopy as analytical tool to study physico-mechanical properties of intestinal cells. Beilstein J Nanotechnol 2015;6:1457-66. 6. Cross SE, Jin YS, Rao J, Gimzewski JK. Nanomechanical analysis of cells from cancer patients. Nat Nanotechnol 2007;2(12):780-3. 7. Sarna M, Wojcik KA, Hermanowicz P, et al. Undifferentiated bronchial fibroblasts derived from asthmatic patients display higher elastic modulus than their non-asthmatic counterparts. PLoS One 2015;10(2):e0116840. 8. Sarna M, Zadlo A, Koczurkiewicz P, Burda K, Sarna T. Malanin Modifies Nanomechanical Properties of Melanoma Cells. The melanocyte and its environment. Bologna, Italy: Medimond; 2012. p. 23-8. 9. Sarna M, Zadlo A, Pilat A, et al. Nanomechanical analysis of pigmented human melanoma cells. Pigment Cell Melanoma Res 2013;26(5):727-30.
332
334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357
M. Sarna et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2016) xxx–xxx
E
D
P
R O O
F
23. Hermanowicz P, Sarna M, Burda K, Gabryś H. AtomicJ: an open source software for analysis of force curves. Rev Sci Instrum 2014;85(6):063703. 24. Sealy RC, Hyde JS, Felix CC, Menon IA, Prota G. Eumelanins and pheomelanins: characterization by electron spin resonance spectroscopy. Science 1982;217(4559):545-7. 25. Sarna T, Burke JM, Korytowski W, Rozanowska M, Skumatz CM, Zareba A, et al. Loss of melanin from human RPE with aging: possible role of melanin photooxidation. Exp Eye Res 2001;76:89-98. 26. Gefen A, Weihs D. Mechanical cytoprotection: a review of cytoskeletonprotection approaches for cells. J Biomech 2016;49(8):1321-9. 27. Vargas-Pinto R, Gong H, Vahabikashi A, Johnson M. The effect of the endothelial cell cortex on atomic force microscopy measurements. Biophys J 2013;105(2):300-9. 28. Wagner OI, Rammensee S, Korde N, Wen Q, Leterrier JF, Janmey PA. Softness, strength and self-repair in intermediate filament networks. Exp Cell Res 2007;313(10):2228-35. 29. Burke JM, Skumatz CM, Irving PE, McKay BS. Phenotypic heterogeneity of retinal pigment epithelial cells in vitro and in situ. Exp Eye Res 1996;62(1):63-73. 30. Booij JC, Baas DC, Beisekeeva J, Gorgels TG, Bergen AA. The dynamic nature of Bruch's membrane. Prog Retin Eye Res 2010;29(1):1-18. 31. Last JA, Liliensiek SJ, Nealey PF, Murphya CJ. Determining the mechanical properties of human corneal basement membranes with atomic force microscopy. Struct Biol 2009;167(1):19-24. 32. Zadlo A, Rozanowska MB, Burke JB, Sarna T. Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit ironinduced peroxidation of lipids. Pigment Cell Res 2007;20:52-60. 33. Luthert PJ. Pathogenesis of age-related macular degeneration. Diagn Histopathol 2011;17(1):10-6. 34. Dugel PU, Zimmer CN. Imaging of melanin disruption in age-related macular degeneration using multispectral imaging. Ophthalmic Surg Lasers Imaging Retina 2016;47(2):134-41. 35. van Rij AM, Jones GT, Hill BG, et al. Mechanical inhibition of angiogenesis at the saphenofemoral junction in the surgical treatment of varicose veins: early results of a blinded randomized controlled trial. Circulation 2008;118(1):66-74.
C
T
10. Sarna M, Zadlo A, Hermanowicz P, Madeja Z, Burda K, Sarna T. Cell elasticity is an important indicator of the metastatic phenotype of melanoma cells. Exp Dermatol 2014;23(11):813-8. 11. Lin JY, Fisher DE. Melanocyte biology and skin pigmentation. Nature 2007;445:843-50. 12. Boulton ME. Studying melanin and lipofuscin in RPE cell culture models. Exp Eye Res 2014;126:61-7. 13. Seagle BL, Rezai KA, Kobori Y, Gasyna EM, Rezaei KA, Norris JR. Melanin photoprotection in the human retinal pigment epithelium and its correlation with light-induced cell apoptosis. Proc Natl Acad Sci U S A 2005;102(25):8978-83. 14. Sarna T. Properties and function of the ocular melanin – a photobiophysical view. J Photochem Photobiol B 1992;12(3):215-58. 15. Campbell M, Humphries P. The blood-retina barrier: tight junctions and barrier modulation. Adv Exp Med Biol 2012;763:70-84. 16. Klaassen I, Van Noorden CJ, Schlingemann RO. Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions. Prog Retin Eye Res 2013;34:19-48. 17. Burke JM, Jaffe GJ, Brzeski CM. The effect of culture density and proliferation rate on the expression of ouabain-sensitive Na/K ATPase pumps in cultured human retinal pigment epithelium. Exp Cell Res 1991;194(2):190-4. 18. Burke JM, Henry MM, Zareba M, Sarna T. Photobleaching of melanosomes from retinal pigment epithelium: I. Effects on protein oxidation. Photochem Photobiol 2007;83(4):920-4. 19. Zareba M, Sarna T, Szewczyk G, Burke JM. Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress. Photochem Photobiol 2007;83(4):925-30. 20. d'Ischia M, Wakamatsu K, Napolitano A, et al. Melanins and melanogenesis: methods, standards, protocols. Pigment Cell Melanoma Res 2013;26(5):616-33. 21. Zadlo A, Burke JM, Sarna T. Effect of untreated and photobleached bovine RPE melanosomes on the photoinduced peroxidation of lipids. Photochem Photobiol Sci 2009;8(6):830-7. 22. Hutter JL, Bechhoefer J. Calibration of atomic-force microscope tips. Rev Sci Instrum 1993;64:1868.
E
358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394
U
N C O
R
R
433
7 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432